The tyrosine phosphatase STEP is a developmental suppressor of synaptogenesis

Striatal-Enriched Protein Tyrosine Phosphatase (STEP) constrains synaptic potentiation by dephosphorylating postsynaptic substrates, but its presynaptic role has remained unclear. Here, we identify a previously unrecognized function of STEP in regulating axonal differentiation and synapse assembly. Genetic and pharmacological manipulation of STEP in vivo and in vitro show that STEP limits presynaptic maturation by restricting synaptic vesicle protein clustering along developing hippocampal axons. Using a reconstituted circuit-on-a-chip we show that loss of presynaptic STEP is sufficient to significantly increase the number of axodendritic synapses. Functional imaging further revealed that the increased synaptic puncta observed in STEP KO neurons actively undergo depolarization-evoked vesicle exocytosis, representing bona fide functional synapses. Multielectrode array recordings reveal that STEP deletion increases neuronal excitability, and network synchrony, hallmarks of enhanced presynaptic efficacy. Mechanistically, these effects reflect sustained phosphorylation of STEP promoting presynaptic assembly and release competence. Importantly, inhibiting STEP also rescues presynaptic differentiation defects in Fmr1 KO neurons, implicating aberrant STEP signaling in Fragile X-associated synaptic pathology. Thus, STEP serves as a phosphatase gatekeeper that restrains presynaptic differentiation and neurotransmission, and its inhibition may offer a therapeutic strategy to correct synaptic deficits in Fragile X Syndrome.